RNA editing of the human herpesvirus 8 kaposin transcript eliminates its transforming activity and is induced during lytic replication.

Human herpesvirus 8 is the etiologic agent associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The K12 RNA, which produces as many as three variants of the kaposin protein, as well as a microRNA, is the most abundant transcript expressed in latent Kaposi's sarcoma-associated herpesvirus infection, and yet it is also induced during lytic replication. The portion of the transcript that includes the microRNA and the kaposin A sequence has been shown to have tumorigenic potential. Genome coordinate 117990, which is within this transcript, has been found to be heterogeneous, primarily in RNAs but also among viral DNA sequences. This sequence heterogeneity affects an amino acid in kaposins A and C and the microRNA. The functional effects of this sequence heterogeneity have not been studied, and its origin has not been definitively settled; both RNA editing and heterogeneity at the level of the viral genome have been proposed. Here, we show that transcripts containing A at position 117990 are tumorigenic, while those with G at this position are not. Using a highly sensitive quantitative assay, we observed that, in PEL cells under conditions where more than 60% of cDNAs derived from K12 RNA transcripts have G at coordinate 117990, there is no detectable G in the viral DNA sequence at this position, only A. This result is consistent with RNA editing by one of the host RNA adenosine deaminases (ADARs). Indeed, we observed that purified human ADAR1 efficiently edits K12 RNA in vitro. Remarkably, the amount of editing correlated with the replicative state of the virus; editing levels were nearly 10-fold higher in cells treated to induce lytic viral replication. These results suggest that RNA editing controls the function of one segment of the kaposin transcript, such that it has transforming activity during latent replication and possibly another, as-yet-undetermined, function during lytic replication.

Transcriptional downregulation of ORF50/Rta by methotrexate inhibits the switch of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 from latency to lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cellular dihydrofolate reductase (DHFR) homologue. Methotrexate (MTX), a potent anti-inflammatory agent, inhibits cellular DHFR activity. We investigated the effect of noncytotoxic doses of MTX on latency and lytic KSHV replication in two KSHV-infected primary effusion lymphoma cell lines (BC-3 and BC-1) and in MTX-resistant BC-3 cells (MTX-R-BC-3 cells). Treatment with MTX completely prevented tetradecanoyl phorbol acetate-induced viral DNA replication and strongly decreased viral lytic transcript levels, even in MTX-resistant cells. However, the same treatment had no effect on transcription of cellular genes and KSHV latent genes. One of the lytic transcripts inhibited by MTX, ORF50/Rta (open reading frame), is an immediate-early gene encoding a replication-transcription activator required for expression of other viral lytic genes. Therefore, transcription of genes downstream of ORF50/Rta was inhibited, including those encoding the viral G-protein-coupled receptor (GPCR), viral interleukin-6, and K12/kaposin, which have been shown to be transforming in vitro and oncogenic in mice. Resistance to MTX has been documented in cultured cells and also in patients treated with this drug. However, MTX showed an inhibitory activity even in MTX-R-BC-3 cells. Two currently available antiherpesvirus drugs, cidofovir and foscarnet, had no effect on the transcription of these viral oncogenes and ORF50/Rta. MTX is the first example of a compound shown to downregulate the expression of ORF50/Rta and therefore prevent viral transforming gene transcription. Given that the expression of these genes may be important for tumor development, MTX could play a role in the future management of KSHV-associated malignancies.